We seek new, medically relevant insights into the biology of red blood cell (RBC) formation (erythropoiesis). In erythroid precursors, the ubiquitin proteasome system (UPS) identifies and eliminates endogenous proteins that become unnecessary or potentially deleterious during progressive maturation. The UPS also functions as a protective mechanism to eliminate toxic proteins that accumulate in RBC disorders, as we and others have demonstrated for ? thalassemia, a common anemia caused by imbalanced hemoglobin synthesis. While the UPS is believed to be critical for erythropoiesis, very little is known regarding the specific molecules involved. Large-scale genome wide association studies (GWAS) of human populations have identified numerous UPS components predicted to regulate erythropoiesis. We combined these GWAS with global transcriptome analyses to identify several potentially important UPS proteins expressed in RBC precursors. One interesting candidate that we have studied in depth is Trim58, a protein that marks other proteins for degradation and has also been implicated by GWAS to regulate the formation of platelets. We showed that Trim58 deficient RBC precursors exhibit faulty maturation, including impaired ability to expel the nucleus, a key step in mammalian erythropoiesis. Preliminary studies indicate that Trim58 facilitates enucleation by eliminating dynein, a molecular motor complex with multiple essential functions in virtually all other cell types. We will perform biochemical studies of purifed proteins and genetic manipulations of cultured RBCs to examine the mechanisms by which Trim58 degrades dynein and how this facilitates RBC precursor enucleation. To investigate potential dynein independent functions of Trim58, we will perform proteomic studies to identify its additional degradation targets (Aim 1). To examine Trim58 functions in vivo, we will ablate the gene in mice and determine the consequences on RBC and platelet formation at baseline and after exposure to various physiological stresses (Aim 2). Finally, we will use short hairpin RNAs to suppress the expression of additional GWAS-identified UPS candidates in cultured primary erythroid precursors and determine how this affects their maturation (Aim 3). Our studies aim to elucidate new pathways that promote erythropoiesis through regulated protein degradation. By altering these pathways through drugs or genetic manipulation, it should be possible to enhance ongoing efforts to generate RBCs in vitro for transfusion therapies and to treat various blood diseases caused by dysregulated erythropoiesis. More generally, our planned investigations synergize with GWAS to better understand how genetic variation influences medically relevant phenotypes.